Papillomaviruses (PVs) infect the epithelia of animals and man where they generally induce benign proliferation at the site of infection. However, there is a strong association between malignant progression of human genital lesions and certain HPV types, most frequently HPV 16. We have generated virus-like particles (VLPs) for HPV 16 and other PVs that consist of the L1 major capsid protein or L1 plus L2, the minor capsid protein. Parenteral injection of purifed VLPs induced high titers of neutralizing antibodies and protection from experimental challenge in animal models. Based upon these results, we are currently organizing a clinical trial of an HPV16 VLP vaccine. In addition, we are developing alternative vaccine candidates. To increase the therapeutic potential of a VLP-based vaccine, we have incorporated non-structural HPV proteins into the VLPs as L2 fusion proteins. Vaccination with an HPV16 E7 chimeric VLP generated a CD8 restricted T cell response that protected mice from tumor challenge using an E7 expressing tumor line. To increase mucosal antibody responses and reduce the expense of vaccine production, we have generated L1 recombinant Salmonella. Contrary to expectations, VLPs assembled in the bacteria and intranasal instillation of the live recombinants in mice produced high titers of neutralizing antibodies. In contrast to parenteral inoculation of VLPs, this protocol also elicited secretory IgA in the female genital tract. Based upon our recent development of a procedure to generate infectious PVs in vitro, we have begun an analysis to determine the mechanism of virion assembly. Although L1 alone self-assembles into VLPs, the L2 minor capsid proteins and the E2 transcription/replication factor were also required to encapsidate the genome and generate infectious virions. A series of experiments examining the subcellular localization of the virion components and E2 supported a model for virion assembly that is based on the ability of L2 to recruit the other essential viral components to distinct nuclear structures previously designated PODs. The specificity of viral genome encapsidation is likely to result from the high affinity binding of E2 to specific viral genome sequences coupled with protein/protein interactions between E2 and an L2/POD complex. Using our previously validated HPV16 VLP-based ELISA, we have compared the virion antibody respose in men and women, examined the relationship of this response to cervical disease progression and regression, and quantitated the local genital mucosal antibody response to HPV infection. In addition, we have use the assay to confirm and extend our previous observations that a serological response to HPV16 is associated with an increased risk of cervical, vulvar and esophageal cancer.